Abstract:

DUF1218 is a land plant-specific innovation and has previously been shown to be associated
with cell wall biology, vasculature patterning and abiotic/biotic stress response. The
Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are
preferentially expressed in the secondary cell wall depositing inflorescence stems. To further
our understanding of the roles of DUF1218-containing proteins in secondary cell wall
biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL
LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional
redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to
MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins
are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2,
and independent overexpression lines showed no significant differences in plant growth or
changes in total lignin content relative to wild-type (WT) control plants. However, the double
homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in
rosette fresh weight and stem height relative to the WT control at four weeks and six weeks,
respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content
(by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative
to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin
biosynthesis, and these proteins appear to function redundantly.

Description:

S1 Fig. Phylogenetic and bioinformatics analysis of all members of the Arabidopsis domain
of unknown function 1218 (DUF1218) family, and expression profiling of the candidate
members, MODIFYING WALL LIGNIN-1 (MWL-1, At1g31720) and MWL-2
(At4g19370). (A) Neighbor-joining phylogenetic tree of Arabdiopsis DUF1218-containing
proteins. ClustalW was used to align protein sequences from TAIR and the alignment thereafter
used to construct the tree using p-distance and pairwise deletion with 1000 bootstrap replicates
in MEGA5 [16]. Prediction of subcellular localization, signal peptide and number of
transmembrane domains was done using SUBA3 [31], Signal-3L [18] and TMHMM[19]
respectively, with default settings. Highlighted in pink are the related MWL-1 and 2 sequences.
(B) Arabidopsis expression profiles for MWL-1 and MWL-2 across different tissues during
development, exctracted from The Bio-Analytic Resource for Plant Biology (http://bar.
utoronto.ca/welcome.htm) [20]. Preferential expression is seen at distinct developmental
stages, however, there is overlap in the secondary cell wall depositing, 2nd internode region.
(DOCX)

S2 Fig. Gene ontology enrichment of MWL-1 top 300 co-expressed genes in Arabidopsis.
Co-expressed genes were extracted from ATTED-II [10]. GO-full was conducted in Cytoscape
2.8.2 [22] using BiNGO 2.44 [21], while overrepresentation summary enrichment was performed
with the REVIGO server [23].
(DOCX)

S3 Fig. Phenotypic analysis of At1g31720 (MWL-1) single T-DNA knockout line mutants
and MWL-1 overexpression lines. (A) RT-PCR detection of endogenous MWL-1 transcript in
the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative
RT-PCR analysis of MWL-1 overexpression lines 1 to 3 showing detection of MWL-1 transgene
in the transgenic lines. Actin2 was used as a control gene and RT-PCR was performed on
cDNA from stem tissue. Actin2 and MWL-1 gene-specific oligonucleotide sequences can be
found in S1 Table. Rosette size (C) and mass (D) of MWL-1 single T-DNA knockout line and
overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative
(F) stem length of MWL-1 single T-DNA knockout line and overexpression lines relative
to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66.
Error bars indicate the standard error. Scale bar, 3 cm. Based on a two-tailed Student’s t-test
(P-value 0.05) no significant differences were seen in the growth and development of the single
mutant and transgenic OE lines in comparison to the WT controls.

S4 Fig. Phenotypic analysis of At4g19370 (MWL-2) single T-DNA knockout line mutants
and MWL-2 overexpression lines. (A) RT-PCR detection of endogenous MWL-2 transcript in
the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative
RT-PCR analysis of MWL-2 overexpression lines 1 to 3 showing detection of MWL-2 transgene
in the transgenic lines except for OE1 which could be indicative of positional effect (position in
the genome), or co-suppression dominant repression. Actin2 was used as a control gene and
RT-PCR was performed on stem tissue. Actin2 and MWL-2 gene-specific oligonucleotide
sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-2 single T-DNA
knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative
(E) and quantitative (F) stem length of MWL-2 single T-DNA knockout line and overexpression
lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative
stem length n = 66. Error bars indicate the standard error while significant difference from the
WT based on a two-tailed Student’s t-test (P-value 0.05) is indicated by . Scale bar, 3 cm. No significant differences were seen in the growth and development of the single mutant and
transgenic OE lines in comparison to the WT controls except for OE-Line 2.
(DOCX)